Crystal structure of the human symplekin-Ssu72-CTD phosphopeptide complex.
Crystal structure of the human symplekin-Ssu72-CTD phosphopeptide complex.
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Symplekin（酵母中的Pta 1）是大蛋白复合物中的支架，其是真核信使RNA前体（pre-mRNAs）的3′-末端切割和多腺苷酸化所需的，并且还参与RNA聚合酶II（Pol II）的转录起始和终止。Symplekin介导许多不同蛋白质之间的相互作用，虽然其功能的分子基础尚不清楚。在这里，我们报告的晶体结构在2.4 nm分辨率的N-末端结构域（残基30-340）的人symplekin（Symp-N）的三元复合物与Pol II C-末端结构域（CTD）Ser 5磷酸酶Ssu 72和CTD Ser 5磷酸肽。symplekin的N-末端结构域具有ARM或HEAT折叠，具有以弧形排列的七对反平行α-螺旋。Ssu 72的结构与低分子量的磷酸酪氨酸蛋白磷酸酶有一些相似之处，尽管Ssu 72具有独特的活性位点景观以及C-末端的额外结构特征，这对于与symplekin的相互作用很重要。Ssu 72与symplekin的凹面结合，在这个界面上的工程突变可以消除两种蛋白质之间的相互作用。CTD肽结合在Ssu 72的活性位点中，出乎意料地具有顺式构型的pSer 5-Pro 6肽键，这与所有其他已知的CTD肽构象形成对比。虽然Ssu 72的活性位点距离symplekin的界面约25 μ m，但我们发现symplekin N-末端结构域在体外刺激Ssu 72 CTD磷酸酶活性。此外，symplekin的N-末端结构域在体外抑制聚腺苷酸化，但重要的是，只有当耦合到转录。由于具有催化活性的Ssu 72克服了这种抑制作用，我们的研究结果证明了哺乳动物Ssu 72在转录偶联前mRNA 3′端加工中的作用。
Symplekin (Pta1 in yeast) is a scaffold in the large protein complex that is required for 3′-end cleavage and polyadenylation of eukaryotic messenger RNA precursors (pre-mRNAs), and also participates in transcription initiation and termination by RNA polymerase II (Pol II). Symplekin mediates interactions among many different proteins in this machinery, although the molecular basis for its function is not known. Here we report the crystal structure at 2.4 Å resolution of the N-terminal domain (residues 30–340) of human symplekin (Symp-N) in a ternary complex with the Pol II C-terminal domain (CTD) Ser5 phosphatase Ssu72 and a CTD Ser5 phosphopeptide. The N-terminal domain of symplekin has the ARM or HEAT fold, with seven pairs of anti-parallel α-helices arranged in the shape of an arc. The structure of Ssu72 has some similarity to that of low-molecular-weight phosphotyrosine protein phosphatase, although Ssu72 has a unique active site landscape as well as extra structural features at the C-terminus that is important for interaction with symplekin. Ssu72 is bound to the concave face of symplekin, and engineered mutations in this interface can abolish interactions between the two proteins. The CTD peptide is bound in the active site of Ssu72, unexpectedly with the pSer5-Pro6 peptide bond in the cis configuration, which contrasts with all other known CTD peptide conformations. While the active site of Ssu72 is about 25 Å away from the interface with symplekin, we found that the symplekin N-terminal domain stimulates Ssu72 CTD phosphatase activity in vitro. Furthermore, the N-terminal domain of symplekin inhibits polyadenylation in vitro, but importantly only when coupled to transcription. As catalytically active Ssu72 overcomes this inhibition, our results demonstrate a role for mammalian Ssu72 in transcription-coupled pre-mRNA 3′-end processing.
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影响因子：
5.3
作者：
Reyes-Reyes, Mariela;Hampsey, Michael
通讯作者：
Hampsey, Michael
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
56.9
作者：
HENDRICKSON, WA
通讯作者：
HENDRICKSON, WA
影响因子：
64.8
作者：
Hirose, Y;Manley, JL
通讯作者：
Manley, JL
影响因子：
5.6
作者：
Kennedy, Sarah A.;Frazier, Monica L.;Steiniger, Mindy;Mast, Ann M.;Marzluff, William F.;Redinbo, Matthew R.
通讯作者：
Redinbo, Matthew R.